Characterization of Β-Estradiol 3-Glucuronidation in Rat Brain

1556 Biol. Pharm. Bull. 40, 1556–1560 (2017) Vol. 40, No. 9 Note

Characterization of β-Estradiol 3-Glucuronidation in Rat Brain Yuki Asai, Yukiko Sakakibara, Haruka Onouchi, Masayuki Nadai, and Miki Katoh* Department of Pharmaceutics, Faculty of Pharmacy, Meijo University; 150 Yagotoyama, Tempaku-ku, Nagoya 468– 8503, Japan. Received January 27, 2017; accepted May 10, 2017

β-Estradiol is conjugated by uridine 5 -diphosphate-glucuronosyltransferase (UGT) 1A to 3-glucuronide in the human liver. UGT1A has been found in the brain; therefore, UGT1A may be involved in β-estradiol 3-glucuronidation in the brain. In the present study, we aimed to characterize the β-estradiol 3-glucuronidation reaction in the rat brain. β-Estradiol 3-glucuronidation was detected in eight rat brain regions (cerebellum, frontal cortex, parietal cortex, piriform cortex, hippocampus, medulla oblongata, striatum, and thala-

mus). β-Estradiol 3-glucuronidation in the cerebellum was fitted to the Hill equation (S508.0 µM, n1.1). In inhibition experiments, β-estradiol 3-glucuronidation was inhibited to 73.6% in the cerebellum by 50 µM bilirubin, whereas it was reduced to 20.5% with 5 µM bilirubin in the liver. Unlike in the liver, Ugt1a1 may not be the main isoform catalyzing this glucuronidation in the brain. Serotonin and acetaminophen at 10 mM inhibited glucuronidation to 1.17 and 25.5%, respectively, in the cerebellum. In induction experiments, the administration of β-naphthoflavone, carbamazepine, and phenobarbital did not increase β-estradiol 3-glucuronidation in the brain except for phenobarbital in the striatum. In addition, β-estradiol 3-glucuronidation was not correlated with serotonin or acetaminophen glucuronidation in the brain, suggesting that Ugt1a6 and Ugt1a7 are not major isoforms of β-estradiol 3-glucuronidation in the rat brain. In the present study, although we were unable to identify the isoform responsible for β-estradiol 3-glucuronidation, we confirmed that β-estradiol could be metabolized to glucuronide in the brain under a different metabolic profile from that in the liver. Key words β-estradiol; uridine 5′-diphosphate-glucuronosyltransferase; brain; enzyme kinetics; inhibition; induction

Uridine 5′-diphosphate-glucuronosyltransferase (UGT) is a Ugt1a isoforms. The purpose of this study was to character- phase II drug-metabolizing enzyme that catalyzes the conju- ize the β-estradiol 3-glucuronidation reaction in the rat brain. gation of many endogenous compounds such as neurosteroids1) We conducted kinetic, inhibition, and induction studies of this and neurotransmitters.2,3) While UGT1A is expressed mainly reaction in the rat brain. in the liver,4) it is also found in extrahepatic tissues including the small intestine5) and kidney.6) Recent reports showed that MATERIALS AND METHODS constitutive expression of UGT1A mRNA was detected in the brain,7,8) neurons,9) astrocytes,10) and microglia,11) implying Materials Acetaminophen (APAP) was purchased from that UGT1A may be involved in modulating the concentra- Nacalai Tesque (Kyoto, Japan). Alamethicin, APAP β-D- tions of neurosteroids and neurotransmitters in the central glucuronide, β-estradiol, β-estradiol 3-(β-D-glucuronide) nervous system. sodium salt, serotonin hydrochloride, and uridine 5′-diphos- A female sex steroid, β-estradiol is synthesized by aro- phoglucuronic acid trisodium salt were obtained from Sigma- 12) matase from testosterone in the ovary or testis and is also Aldrich (St. Louis, MO, U.S.A.). Serotonin β-D-glucuronide 13) produced in the brain. β-Estradiol has a protective effect in and serotonin-d4 β-D-glucuronide were purchased from To- neuronal cells against oxidative stress during ischemic brain ronto Research Chemicals (Toronto, Canada). Carbamazepine injury14) and Alzheimer’s disease.15) The major metabolic (CBZ), β-naphthoﬂavone (BNF), and phenobarbital (PB) were pathway of β-estradiol involves glucuronidation at 3-hydroxy obtained from Wako Pure Chemical Industries, Ltd. (Osaka, position.16) β-Estradiol 3-glucuronide is formed by UGT1A1, Japan). All other chemicals and solvents were of the highest UGT1A3, UGT1A8, UGT1A10, UGT2A1, and UGT2A2 in hu- commercial grade available. mans.17) Considering that β-estradiol 3-glucuronide has been Preparation of Brain and Liver Microsomes The pres- found in the mouse brain,18) β-estradiol could be metabolized ent study was approved by the Institution Animal Care and by UGTs in the brain, as glucuronide, with its high water solu- Use Committee of Meijo University. Brains and livers were bility, generally does not easily penetrating the blood-brain removed from eight-week-old male Sprague-Dawley rats barrier. However, the kinetics of β-estradiol 3-glucuronidation (Japan SLC, Hamamatsu, Japan). The cerebellum, frontal in the brain remain to be elucidated. cortex, parietal cortex, piriform cortex, hippocampus, me- In terms of the metabolism of β-estradiol in rats, a previous dulla oblongata, striatum, and thalamus were isolated from the study reported that β-estradiol 3-glucuronidation occurred in brain. Pooled rat microsomes from brain regions (n=8) and recombinant Ugt1a1 and Ugt1a719) but not in those recombi- livers (n=3) were prepared as described previously.21) nant Ugt2b1, Ugt2b2, or Ugt2b3.1) It was not observed in the β-Estradiol 3-Glucuronidation in Rat Brain Regions liver microsomes of Gunn rats,20) suggesting that, similar to β-Estradiol 3-glucuronide levels were determined using a humans, β-estradiol 3-glucuronidation is catalyzed mainly by method described previously22) with slight modiﬁcations. The

* To whom correspondence should be addressed. e-mail: [email protected] © 2017 The Pharmaceutical Society of Japan Vol. 40, No. 9 (2017) Biol. Pharm. Bull. 1557 incubation mixture contained 50 mM Tris (hydroxymethyl) aminomethane–HCl (pH 7.4), 5 mM MgCl2, 25 µg/mL alamethicin, 3 mM uridine 5′-diphosphoglucuronic acid, 5 µM β-estradiol, and microsomal proteins (brain: 0.5 mg/mL or liver: 0.01 mg/mL). The reaction mixtures were incubated at 37°C at 24 h (brain) or 10 min (liver). β-Estradiol 3-glucuronide was quantified by LC-MS/MS. The mobile phase was 0.15% ammonium and methanol (75 : 25, v/v) at a flow rate of 0.2 mL/min. The detection limit for β-estradiol 3-glucuronide was 4.2 fmol. The quantification limit in the reaction mixture was 21 nM with a coefficient of variation of less than 10%. Kinetic Analysis of β-Estradiol 3-Glucuronidation in Rat Cerebellum The concentration of β-estradiol ranged from 0.2 to 200 µM. Kinetic analysis was performed using the KaleidaGraph computer program (Synergy Software, Read- ing, PA, U.S.A.). The following equation was applied for Hill n n n kinetic models: V=Vmax×S /(S50 +S ), where V is the velocity of the reaction, Vmax is the maximum velocity, S is the sub- Fig. 1. β-Estradiol 3-Glucuronidation in Regions of the Rat Brain and strate concentration, S50 is the substrate concentration at the Liver half-Vmax, and n is the Hill coefficient. Maximum clearance (CL ) was calculated as V ×(n−1)/[S ×n (n−1)1/n] for the The concentration of β-estradiol was 5 µM. Each column represents the max max 50 mean±S.D. of three independent determinations. Hill kinetic. Inhibition Study for β-Estradiol 3-Glucuronidation in Rat Cerebellum and Liver The inhibition study was conducted at 5 µM β-estradiol. Bilirubin (5–50 µM), serotonin (1–10 mM), and APAP (1–10 mM) were glucuronidated mainly by Ugt1a1,23) Ugt1a6,21) and Ugt1a6 and Ugt1a7,24) respectively, which were used as the Ugt inhibitors. β-Estradiol 3-glucuronidation was analyzed in the presence of each Ugt inhibitor. Induction Study of β-Estradiol 3-Glucuronidation in Rat Brain Regions and Liver Eight-week-old male Sprague- Dawley rats were treated intraperitoneally once daily for 7 d with 80 mg/kg BNF, 100 mg/kg CBZ, or 80 mg/kg PB. For controls, rats were treated with corn oil (control for BNF and CBZ) or saline (control for PB). β-Estradiol 3-glucuronidation Fig. 2. Eadie–Hofstee Plot for β-Estradiol 3-Glucuronidation in the Rat was measured using microsomes from eight brain regions and Cerebellum livers, as mentioned above. For correlation analysis, serotonin The β-estradiol concentrations ranged from 0.2 to 200 µM. The points represent glucuronidation and APAP glucuronidation were also exam- the mean of three independent determinations. ined. Serotonin glucuronidation was determined by a previous method21) with slight modification. APAP glucuronidation was 0.16 nL/min/mg protein, respectively. In some previous stud- performed according to a method described previously.25) ies, β-estradiol 3-glucuronidation in the liver was also fitted

Statistical Analysis Statistical analysis of the experimen- to the Hill equation with an S50 of 16–33 µM and a Hill coef- tal data was performed using Student’s paired t-test with the ficient of 1.8–2.2.28–30) This suggests that the Ugt isoforms KaleidaGraph computer system. catalyzing β-estradiol 3-glucuronidation in the brain may be different from those in the liver. RESULTS AND DISCUSSION A previous study using recombinant rat Ugt1a isoforms reported that Ugt1a1 and Ugt1a7 (but not Ugt1a2 or Ugt1a3) β-Estradiol 3-glucuronidation was detected in all eight were involved in β-estradiol 3-glucuronidation.19) 1-Naphthol brain regions (Fig. 1). There were, however, regional differ- glucuronidation, which is catalyzed mainly by Ugt1a6, was ences in β-estradiol 3-glucuronidation. The highest activity inhibited by β-estradiol in rat ovarian cells.31) among the eight brain regions was observed in the hippocam- For elucidation of the Ugt1a isoforms responsible for pus (0.99 fmol/min/mg protein). The β-estradiol level is known β-estradiol 3-glucuronidation in the rat brain, the inhibi- to be associated with hippocampus-dependent memory.26) A tion of this glucuronidation by typical Ugt1a inhibitors was β-estradiol-generating enzyme, aromatase, is expressed at determined (Fig. 3). β-Estradiol 3-glucuronidation in the cer- high levels in the hippocampus CA1–CA3 regions.27) Because ebellum was reduced to 73.6% in the presence of bilirubin at UGT is an enzyme involved in the degradation of β-estradiol, 50 µM. In contrast, in the liver, β-estradiol 3-glucuronidation high β-estradiol glucuronidation activity in the hippocampus was reduced to 20.5% by bilirubin at 5 µM. The inhibitory would serve to maintain β-estradiol levels in the hippocampus. potency of the Ugt1a1 inhibitor bilirubin was much weaker β-Estradiol 3-glucuronidation in the cerebellum was fitted in the cerebellum than in the liver. Although Ugt1a1, which is to the Hill equation (Fig. 2). The S50, Vmax, Hill coefficient, expressed at the highest mRNA levels among hepatic Ugt1a 32) and CLmax were 8.0 µM, 1.30 fmol/min/mg protein, 1.1, and isoforms, accounts mainly for β-estradiol 3-glucuronidation 1558 Biol. Pharm. Bull. Vol. 40, No. 9 (2017)

Fig. 3. Inhibitory Effects of Bilirubin (A), Serotonin (B), and APAP (C) on β-Estradiol 3-Glucuronidation in the Rat Cerebellum and Liver

β-Estradiol 3-glucuronidation was determined at 5 µM β-estradiol in the presence of UGT inhibitors (bilirubin: 5–50 µM, serotonin: 1–10 mM, APAP: 1–10 mM). Residual activities were calculated by setting the activity in the absence of inhibitors at 100%. Each data point represents the mean of three independent determinations.

Fig. 4. Changes in β-Estradiol 3-Glucuronidation Following Treatment with BNF, CBZ, and PB in Regions of the Rat Brain and Liver

The concentration of β-estradiol was 5 µM. Each column represents the mean±S.D. of three independent determinations. * p<0.05, ** p<0.01, *** p<0.001 compared with controls. in the liver, it may be the minor isoform catalyzing this gluc- bon receptor, pregnane X receptor, and constitutive androstane uronidation in the brain. The inhibitory effects of serotonin receptor, respectively.34) In the eight brain regions (Fig. 4), and APAP on β-estradiol 3-glucuronidation in the cerebellum BNF, CBZ, and PB did not increase β-estradiol 3-glucuronida- were similar to those in the liver. The residual activities in tion, except for a 1.3-fold increase in the striatum with PB. the cerebellum were 1.17 and 25.5% in the presence of 10 mM UGT1A is responsible for the conjugation of neurotransmit- 2) 3) serotonin and 10 mM APAP, respectively. Constitutive mRNA ters such as serotonin and dopamine. The levels of some expression levels of Ugt1a6 and Ugt1a7 are approximately Ugt1a isoforms that catalyze β-estradiol 3-glucuronidation 16- and 13-fold higher, respectively, than that of Ugt1a1 in the were elevated by PB in the striatum, where serotonergic and brain.8) Thus, the results from the inhibition study showed that dopaminergic nerve terminals exist; therefore, the concentra- Ugt1a6 and Ugt1a7 might be the primary isoforms involved in tions of these neurotransmitters may change locally. However, β-estradiol 3-glucuronidation in the rat brain. β-estradiol 3-glucuronidation was elevated 1.3- and 1.6-fold in Ugt1a6 and Ugt1a7 mRNAs in the rat brain have been re- the liver following CBZ and PB administration, respectively. ported to be induced by treatment with BNF25) and PB33) at β-Estradiol 3-glucuronidation was unchanged by treatment the same dosages as those in the present study. If β-estradiol with BNF. Since Ugt1a1 is a target gene of pregnane X recep- 3-glucuronidation is catalyzed mainly by Ugt1a6 and Ugt1a7, tor and constitutive androstane receptor,35) a statistically sig- this glucuronidation will be increased by the Ugt inducers in niﬁcant increase in β-estradiol 3-glucuronidation in the liver vivo. The present study used BNF, CBZ, and PB as the Ugt in- presumably resulted from Ugt1a1 induction following CBZ ducers, stimulating the nuclear translocation of aryl hydrocar- and PB administration (Fig. 4). Ugt1a6 mRNA was increased Vol. 40, No. 9 (2017) Biol. Pharm. Bull. 1559

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